Expression of MSLN in pancreatic cancer cell lines and pancreatic cancer tissues. A. MSLN protein expression in pancreatic cancer cell lines and human pancreatic ductal epithelium (PHDE) cells by Western blot analysis. B. MSLN mRNA in pancreatic tissues detected by real-time RT-PCR. Twenty-one paired tumor and adjacent normal tissues were studied. Relative mRNA concentrations of MSLN were normalized to that of GAPDH and presented as 2^(Ct_[GAPDH] - Ct_[MSLN]). C. Immunohistochemical staining of MSLN expression in pancreatic cancer tissues.

Effects of MSLN overexpression in MIA PaCa-2 cells on cell proliferation and migration. A. MSLN expression in stable cells by Western blot analysis. B. Cell proliferation by MTS assay. Cells were seeded in 96-well plates (2 × 10³ cells/well) and serum-starved for 24 h before changing to the growth medium with 2% FBS. Absorbance was recorded on day 6 after release from starvation at 490 nm. C. Cell migration by modified Boyden chamber assay. Stable cells were trypsinized and resuspended in growth medium (10⁵ cells/500 μL) and added into the upper compartment of a migration chamber for 24 h. The cells were stained with Calcein-AM. The mean fluorescence reading after scraping of the cells at the top was divided by the reading before removal of the top cells, and the ratio was plotted. D. Cell migration by wound-healing assay. Confluent stable cells were scraped with a pipette tip and cultured in DMEM for 3 days. Pictures were taken daily. E. Cell-cycle analysis by flow cytometry. Confluent cells were serum-starved (0% FBS) for 24 h before changing to the growth medium with 2% FBS. Cells were collected at 4 h and 8 h, permeabilized, stained with propidium iodine (PI), and analyzed with flow cytometry. **p < 0.001.

Effects of silencing of MSLN expression in BxPC-3 cells on cell proliferation and migration. A. MSLN expression in stable cells by Western blot analysis. B. Cell proliferation by MTS assay. Cells were seeded in 96-well plates (2 × 10³ cells/well) and serum-starved (0% FBS). Cell growth was assessed with the MTS assay on day 6 after releasing from starvation. C. Cell migration by modified Boyden chamber assay with calcein-AM labeling. **p < 0.001.

Effects of MSLN on pancreatic cancer growth in the xenograft nude mouse model. A. Subcutaneous tumor volume of MIA-MSLN cells. MIA-MSLN or MIA-V cells (2 × 10⁶) were subcutaneously inoculated into nude mice (10 mice per treatment group). Tumor size was measured weekly for 4 weeks. **p < 0.01. B. Subcutaneous tumor volume of BxPC-siMSLN cells. 2 × 10⁶ BxPC-siMSLN cells were injected into the right flank of nude mice (eight per treatment group). Tumor size was measured weekly for 6 weeks. **p < 0.01. C. Orthotopic tumor volume of MIA-MSLN cells. 2×10⁶ MIA-MSLN or MIA-V cells were implanted into the body of pancreases in nude mice (10 in each group). The mice were euthanized at different times. Orthotopic tumor volume was measured. Tumor volume in mice given MIA-MSLN inoculation was significantly higher than that in mice given MIA-V inoculation (**p < 0.01). D. Survival rate of nude mice with orthotopically inoculated tumors. 2 × 10⁶ MIA-MSLN or MIAV cells were implanted into the body of pancreases in nude mice (10 in each group). Death was recorded up to 4 weeks after tumor inoculation. Survival of mice with MIA-MSLN inoculations was substantially lower than that of mice with MIA-V inoculations (p<0.00001).

Effects of therapeutic vaccine against MSLN on pancreatic cancer growth and pathogenesis in the orthotopic mouse model. A. Effects of VLP-hMSLN vaccination on orthotopic tumor growth in mice. 5 × 10⁵ Panc02 cells were implanted into the body of pancreases of C57BL/6J wild-type mice. On day 3, 50 μg VLP-hMSLN, VLP-Gag, or PBS was injected intraperitoneally (10 mice in each group); this was followed on day 7 and day 14 with two 100 μg VLP-hMSLN, VLP-Gag, or PBS boosts. Tumor growth was analyzed 4 weeks after implantation. Photo images show a representative tumor size in each group at the same distance. The bar graph shows the quantitation of tumor mass in each group. **p<0.01. B. Effects of VLP-hMSLN vaccination on the survival of mice. The same groups of animals as in Fig. 5A were studied. Death was recorded up to 9 weeks after tumor inoculation. Survival of VLP-hMSLN-vaccinated mice was substantially greater than that of VLP-Gag- or PBS-treated mice (p=0.006). C. Effects of VLP-hMSLN vaccination on serum anti-MSLN antibody production. The same groups of animals as in Fig. 5A were studied. Two weeks after the final VLP boost, serum samples were individually collected, and titers of IgG against MSLN were determined by ELISA. Serum anti-MSLN antibodies in the VLP-MSLN-vaccinated mice were substantially increased compared with those in VLPGag- or PBS-treated mice. **p < 0.01 compared with the VLP-Gag group. D. Effects of VLP-hMSLN vaccination on MSLN-specific IFN-γ-producing cells by ELISPOT assay. The same groups of animals as in Fig. 5A were studied. Two weeks after the final VLP boost, mouse spleen cells were collected and stimulated with recombinant vaccinia virus expressing MSLN in vitro. Spleen IFN-γ-producing T cells in the VLP-MSLN-vaccinated mice were substantially increased compared with those in VLP-Gag- or PBS-treated mice. **p < 0.01 compared with the VLP-Gag group. E. Increased MSLN-specific CTL activity elicited by VLP-hMSLN immunization. Three groups of naïve mice and three groups of Panc02 implanted mice were immunized with PBS, VLP-Gag, or VLP-hMSLN at days 3, 7, and 14. At 28 days post immunization, splenocytes isolated from PBS, VLP-Gag, or VLP-hMSLN immunized groups were stimulated with an MSLN-specific peptide at 2 μM for five days to generate effector cells. EL-4 cells pulsed with MSLN-specific peptide (2 μM) for 1 h were used as target cells. Different effector-to-target cell ratios (81:1, 27:1, 9:1, 3:1, and 1:1) were mixed for 4 h. The specific lysis of target cells was measured by LDH release and calculated using formula in the Cytotox 96 assay kit (Promega, Madison, WI). Data are presented as mean ±standard deviation. The data represent one of five independent experiments with different mice. **p<0.01 .

Reduced numbers of Tregs in VLP-hMSLN-vaccinated mice. About 5 × 10⁵ Panc02 cells were implanted into the body of pancreases of C57BL/6J wild-type mice. On day 3, 50 μg VLP-hMSLN, VLPGag, or PBS was injected intraperitoneally (10 mice in each group); this was followed on day 7 and day 14 with two 100-μg VLP-hMSLN, VLP-Gag, or PBS boosts. Two weeks after the final VLP boost, mouse splenocytes were collected and stained for CD4, CD25, and Foxp3 expression by flow cytometry (A.); or mouse Panc02 tumor sections were stained with Foxp3 Ab to visualize number of Treg cells (red cells) in tumors with PBS, VLP-Gag, or VLP-hMSLN immunization (B.). The number of Tregs was lower in VLP-hMSLN vaccinated mice. All data shown are representatives of three separate experiments.